The Imaging Sciences Laboratory has a major collaborative research effort with the Institutes involving the use of image processing techniques and advanced computational techniques in structural biology to analyze electron micrographs and NMR spectra with the goal of determining macromolecular structures and dynamics. Recent efforts have concentrated on the 3D reconstruction, analysis and interpretation of the structures of icosahedral virus capsids in addition to structure determination and analysis of isolated proteins and nucleic acids. Ongoing research involves analyses of structures related to herpesvirus and papillomairus and other icosahedral virus capsids. Papillomaviruses (e.g. HPV-16) encode two capsid proteins, L1 and L2. The major capsid protein, L1, can assemble spontaneously into a 72-pentamer icosahedral structure that closely resembles native virions. Although the minor capsid protein L2 is not required for capsid formation, it is thought to participate in encapsidation of the viral genome, and plays a number of essential roles in the viral infectious entry pathway. Cryo-electron microscopy and difference 3D reconstruction analysis of purified capsids revealed an icosahedrally-ordered L2-specific density beneath the axial lumen of each L1 capsomer. We have used time-lapse cryo-electron microscopy and image analysis to study the maturation of HPV-16 capsids assembled in 293T cells. The major capsid protein, L1, initially forms a loosely connected procapsid which, under in vitro conditions, condenses over several hours into the more familiar 60 nm-diameter papillomavirus capsid. In this process, the procapsid shrinks by 5% in diameter;its pentameric capsomers change in structure, most markedly in their axial region;and the interaction surfaces between adjacent capsomers are consolidated. These structural changes are accompanied by the formation of disulfide crosslinks that enhance the stability of the mature capsid. At slightly basic pH, the capsids do not achieve compete disulfide bond formation (e.g. maturation). Simply buffering the lysate to more neutral conditions allowed the production of recombinant capsids with >90% disulfide bonds. Cryo-EM with image reconstruction revealed that more fully mature recombinant HPV16 capsids exhibited a much greater degree of regularity compared to HPV16 capsids produced using standard procedures. Their greater regularity allowed us to reconstruct the capsid to higher resolution ( 9), sufficient to allow robust fitting of the L1 crystal structure into the density map. The ability to produce fully mature recombinant capsids should benefit further structural investigations of native HPV capsids. Moreover, fully mature pseudovirions should be viewed as preferred reagents for studies aimed at elucidating the entry pathways used by HPV in the course of natural infections. The C175S mutant, which does not crosslink, shows similar maturation-related structural changes but capsids are significantly larger, under otherwise similar conditions. We conclude that the observed structural size changes facilitates maturation, but crosslink formation is required to lock the capsid into the mature state. These results will submitted for publication. We have also been developing computational tools for the study of the structure and dynamics of biological macromolecules using NMR data. Development of the Xplor-NIH software package for structure determination has continued in the following areas: (a) further development of the Python scripting interface along with extensive documentation;(b) the addition of the ability to refine directly against NMR relaxation data, with the specific application to protein complex structure determination;(c) new tools for incorporating SAXS and SANS data into structure calculations, applied to the Enzyme I protein and Enzyme I/HPr protein complex;(d) application of structure refinement tools using data from Solid-State NMR experiments, including the incorporation of solid state paramagnetic enhancement NMR data and fiber diffraction X-ray data in structure determination.